Filarial nematodes remain a significant health problem worldwide, with over 40 million individuals suffering from diseases associated with infection of these parasites. A major hindrance in the study of the human filariae has been the lack of methods to genetically manipulate these parasites. As a result, little or nothing is known concerning the methods used by the human filariae to regulate their gene expression. In the previous project period, we developed three different methods to transiently transfect B. malayi, one of the human filial parasites. The development of this technology is a significant advancement in our ability to understand filarial parasites and the molecular level, and has allowed us to make the first detailed assessments of transcription and mRNA processing in these organisms. In this proposal, we plan to build upon the advances made in the initial funding period. The overall goals of this project will be to conduct a detailed analysis of promoter function and RNA trans splicing in B. malayi, using our homologous transfection system. We will also build upon the knowledge gained to develop an integrative stable transfection method. To accomplish these overall goals, the following specific aims are proposed: 1. To identify elements interacting with conserved transcriptional factors in the HSP70 core promoter of B. malayi. 2. To determine which portions of the core promoter of the HSPT0 gene are conserved in other B. malayi promoters. 3. To dissect the role of downstream cis acting factors necessary for trans splicing of transgenic pre-mRNAs. 4. To develop a stable transfection system for B. malayi.